Cardiovascular Complications Associated with Diabetes are a Major Public Health Problem.
Diabetes mellitus is an epidemic in the United States (Brownlee, “Biochemistry and Molecular Cell Biology of Diabetic Complications,” Nature 414:813-20 (2001); Nishikawa et al., “Normalizing Mitochondrial Superoxide Production Blocks Three Pathways of Hyperglycaemic Damage,” Nature 404:787-90 (2000); Zimmet et al., “Global and Societal Implications of the Diabetes Epidemic,” Nature 414:782-7 (2001)). Currently 15-17 million adults (5% of the adult population) in the U.S. are affected by Type I and Type II diabetes (Harris et al., “Prevalence of Diabetes, Impaired Fasting Glucose, and Impaired Glucose Tolerance in U.S. Adults. The Third National Health and Nutrition Examination Survey, 1988-1994,” Diabetes Care 21:518-24 (1998); AD Association, “Economic Costs of Diabetes in the U.S. in 2002,” Diabetes Care 26:917-932 (2003)). By the year 2020, the diabetic population is expected to increase by another 44% (AD Association, “Economic Costs of Diabetes in the U.S. in 2002,” Diabetes Care 26:917-932 (2003)). In addition to those with diabetes mellitus, an additional number of people display the metabolic syndrome, with impaired glucose and insulin tolerance and altered vascular reactivity.
The greatest impact of diabetes is on the vascular system (Caro et al., “Lifetime Costs of Complications Resulting From Type 2 Diabetes in the U.S. Diabetes Care 25:476-81 (2002)). Diabetic patients have an increased risk for vascular disease affecting the heart, brain, and peripheral vessels (Howard et al., “Prevention Conference VI: Diabetes and Cardiovascular Disease: Writing Group I: Epidemiology,” Circulation 105:el32-7 (2002)). The relative risk of cardiovascular disease in diabetics is 2-8 times higher than age-matched controls (Howard et al., “Prevention Conference VI: Diabetes and Cardiovascular Disease: Writing Group I: Epidemiology,” Circulation 105:e132-7 (2002)). Diabetes accounts for 180 billion dollars in annual health costs in the U.S., with 85% of this amount attributable to vascular complications (Caro et al., “Lifetime Costs of Complications Resulting From Type 2 Diabetes in the U.S. Diabetes Care 25:476-81 (2002)). Indeed, if macrovascular complications (stroke, MI, TIA, angina) and microvascular complications (nephropathy, neuropathy, retinopathy, wound healing) are considered together, the vast majority of diabetes related healthcare expenditures result from vasculopathies.
One of the Reasons Why Diabetic Patients have Poor Outcomes is Because of Impaired Compensatory Vascular Growth.
The recognition that diabetes impairs survival after ischemic events dates back to the last century and has been independently confirmed by two landmark epidemiologic studies (The Framingham Study and The Diabetes Control and Complications Trial) (Garcia et al., “Morbidity and Mortality in Diabetics in the Framingham Population. Sixteen Year Follow-Up Study,” Diabetes 23:105-11 (1974); TDCaCTR Group, “The Effect of Intensive Treatment of Diabetes on the Development and Progression of Long-Term Complications in Insulin-Dependent Diabetes Mellitus,” N Engl J Med 329:977-86 (1993)). These prospective studies substantiated a relationship between poor glycemic control and decreased survival after myocardial infarction. Of note, these trials demonstrated that in addition to an increased incidence of ischemic episodes (Kannel et al., “Diabetes and Cardiovascular Risk Factors: the Framingham Study,” Circulation; 59:8-13 (1979)), diabetic patients have higher rates of post-infarct complications, such as cardiac failure and secondary ischemic events (Haffner et al., “Mortality From Coronary Heart Disease in Subjects With Type 2 Diabetes and in Nondiabetic Subjects With and Without Prior Myocardial Infarction,” N Engl J Med 339:229-34 (1998); Zuanetti et al., “Influence of Diabetes on Mortality in Acute Myocardial Infarction: Data From the GISSI-2 Study,” J Am Coll Cardiol 22:1788-94 (1993)). These disparities were not due to increased infarct size in the diabetic population (Wilson, “Diabetes Mellitus and Coronary Heart Disease,” Am J Kidney Dis 32:S89-100 (1998)), suggesting that an impairment existed in the compensatory response of the diabetic myocardium. Similar impairments have been described in other diabetic tissues, including the extremities and brain (Uusitupa et al., “5-Year Incidence of Atherosclerotic Vascular Disease in Relation to General Risk Factors, Insulin Level, and Abnormalities in Lipoprotein Composition in Non-Insulin-Dependent Diabetic and Nondiabetic Subjects,” Circulation 82:27-36 (1990); Jude et al., “Peripheral Arterial Disease in Diabetic and Nondiabetic Patients: a Comparison of Severity and Outcome,” Diabetes Care 24:1433-7 (2001); Tuomilehto et al., “Diabetes Mellitus as a Risk Factor for Death From Stroke. Prospective Study of the Middle-Aged Finnish Population,” Stroke 27:210-5 (1996)).
The concept that these impairments result from a poorly adapting diabetic vasculature has both clinical and experimental support. Since angiogenesis and collateral development are the processes that restore blood flow to watershed areas of the heart, the rapid restoration of a normal vascular density in the microvasculature ultimately determines patient outcome following ischemia (Helfant et al., “Functional Importance of the Human Coronary Collateral Circulation,” N Engl J Med 284:1277-81 (1971); Chilian et al., “Microvascular Occlusions Promote Coronary Collateral Growth,” Am J Physiol 258:H1103-11 (1990)). Indeed, the theoretical basis for therapeutic angiogenesis is the belief that augmenting the microvascular network in ischemic and watershed areas of the heart would be beneficial. Clinical as well as experimental studies provide conclusive evidence that diabetes impairs ischemia-driven neovascularization (Abaci et al., “Effect of Diabetes Mellitus on Formation of Coronary Collateral Vessels,” Circulation 99:2239-42 (1999); Tooke, “Microvasculature in Diabetes,” Cardiovasc Res 32:764-71 (1996); Waltenberger, “Impaired Collateral Vessel Development in Diabetes: Potential Cellular Mechanisms and Therapeutic Implications,” Cardiovasc Res 49:554-60 (2001); Yarom et al., “Human Coronary Microvessels in Diabetes and Ischaemia. Morphometric Study of Autopsy Material,” J Pathol 166:265-70 (1992)). In animal studies, diabetic animals demonstrate a decreased vascular density following hindlimb ischemia (Rivard et al., “Rescue of Diabetes-Related Impairment of Angiogenesis By Intramuscular Gene Therapy With Adeno-VEGF,” Am J Pathol 154:355-63 (1999); Taniyama et al., “Therapeutic Angiogenesis Induced By Human Hepatocyte Growth Factor Gene in Rat Diabetic Hind Limb Ischemia Model: Molecular Mechanisms of Delayed Angiogenesis in Diabetes,” Circulation 104:2344-50 (2001); Schatteman et al., “Blood-Derived Angioblasts Accelerate Blood-Flow Restoration in Diabetic Mice,” J Clin Invest 106:571-8 (2000)). Human angiographic studies have demonstrated that diabetic patients have fewer collateral vessels than non-diabetic controls (Abaci et al., “Effect of Diabetes Mellitus on Formation of Coronary Collateral Vessels,” Circulation 99:2239-42 (1999)). Moreover, revascularization via coronary angioplasty, coronary artery bypass surgery, or lower extremity revascularization has a significantly lower success rate in diabetic patients even in the presence of a patient bypass conduit, suggesting the existence of a defect at the microcirculatory level (Kip et al., “Coronary Angioplasty in Diabetic Patients. The National Heart, Lung, and Blood Institute Percutaneous Transluminal Coronary Angioplasty Registry,” Circulation 94:1818-25 (1996); Palumbo et al., “Diabetes Mellitus: Incidence, Prevalence, Survivorship, and Causes of Death in Rochester, Minn., 1945-1970,” Diabetes 25:566-73 (1976); Schwartz et al., “Coronary Bypass Graft Patency in Patients With Diabetes in the Bypass Angioplasty Revascularization Investigation (BART),” Circulation 106:2652-8 (2002); Kip et al., “Differential Influence of Diabetes Mellitus on Increased Jeopardized Myocardium After Initial Angioplasty or Bypass Surgery: Bypass Angioplasty Revascularization Investigation,” Circulation 105:1914-20 (2002)).
TABLE 1Published Studies Supporting Impaired Ischemic Responsiveness in DiabetesStudyType of StudyMajor FindingsAbaci et al(a)ClinicalAngiographic demonstration of decreased collaterals in the hearts ofdiabetic patientsAbaci et al(b)ClinicalCardiac failure is more common following an MI in diabetic patientsAltavilla et al(c)ExperimentalDiabetic mice have less VEGF, less angiogenesis and impaired woundhealing compared to normal miceArora et al(d)ClinicalDiabetics undergoing lower-extremity bypass maintain an impairedvascular reactivity even after successful surgical grafting, highlighting thelimits of surgical interventionsBradley et al(e)ClinicalDiabetic patients have worse survival after an MIChou et al(f)ExperimentalFirst demonstration that myocardial tissue and cells from diabetic animalsexpress less VEGF and its receptorsFrank et al(g)ExperimentalDiabetic mice express much less VEGF RNA and protein in their woundsGoova et al(h)ExperimentalBlockade of the RAGE receptor accelerated wound healing, augmentedVEGF expression, and increased angiogenesis in diabetic miceGuzik et al(i)ClinicalBlood vessels from diabetic patients produce augmented levels ofsuperoxide, a marker/cause of oxidative stressHaffner et al(j)ClinicalDiabetic patients have a greatly increased incidence of experiencing an MIand dying from an MIHiller et al(k)ClinicalEpidemiologic study suggesting that diabetic microangiopathy is greatlyincreased in diabeticsJude et al(l)ClinicalDiabetic patients have an increased incidence, severity, and pooreroutcomes in peripheral arterial disease of the lower extremitiesKip et al(m)ClinicalAngiographic and epidemiologic study demonstrating that diabeticpatients have more diffuse atherosclerotic disease, and worm outcomesafter seemingly successful interventional revascularizationLerman et al(n)ExperimentalFirst demonstration that cells isolated from diabetic animals and patientsproduce attenuated levels of VEGF in hypoxiaMarsh et al(o)ExperimentalMonocytes from diabetic patients without retinopathy express less VEGFin hypoxia compared to monocytes from patients with diabetic retinopathyPartamian et al(p)ClinicalDiabetic patients have increased peri-infarct complications and decreasedlong-term survivalRivard et al(q)ExperimentalDiabetes decreases reactive angiogenesis and tissue survival followinghindlimb ischemiaSchatteman et al(r)ExperimentalAngioblasts from diabetic humans show decreased proliferation anddifferentiation to mature endothelial cells in culture. Also, diabetic micehave less tolerance to hindlimb ischemia than nondiabetic miceTepper et al(s)ExperimentalFirst demonstration that endothelial progenitor cells from diabetic patientsshow decreased function with assays that measure functions important forangiogenesisYarom et al(t)ClinicalAutopsy pathologic study demonstrating that diabetic patients havedecreased ischemia-induced reactive angiogenesis(a)Abaci et al., “Effect of Diabetes Mellitus on Formation of Coronary Collateral Vessels,” Circulation 99: 2239–42 (1999)(b)Abbott et al., The Impact of Diabetes on Survival Following Myocardial Infarction in Men vs Women. Framingham Study,” Jama 260: 3456–60 (1988).(c)Altavilla et al., “Inhibition of Lipid Peroxidation Restores Impaired Vascular Endothelial Growth Factor Expression and Stimulates Wound Healing and Angiogenesis in the Genetically Diabetic Mouse,” Diabetes 50: 667–74 (2001}.(d)Arora et al., “Cutaneous Microcirculation in the Neuropathic Diabetic Foot Improves Significantly But Not Completely After Successful Lower Extremity Revascularization,” J Vasc Surg 35: 501–5 (2002).(e)Bradley et al., “Survival of Diabetic Patients After Myocardial Infarction,” Am J Med 20: 207–216 (1956).(f)Chou et al., “Decreased Cardiac Expression of Vascular Endothelial Growth Factor and its Receptors in Insulin-Resistant and Diabetic States: A Possible Explanation for Impaired Collateral Formation in Cardiac Tissue,” Circulation 105: 373–9 (2002).(g)Frank et al., “Regulation of Vascular Endothelial Growth Factor Expression in Cultured Keratinocytes. Implications for Normal and Impaired Wound Healing,” J Biol Chem 270: 12607–13 (1995).(h)Goova et al., “Blockade of Receptor for Advanced Glycation End-Products Restores Effective Wound Healing in Diabetic Mice,” Am J Pathol 159: 513–25 (2001).(i)Guzik et al., “Mechanisms of Increased Vascular Superoxide Production in Human Diabetes Mellitus: Role of NAD(P)H Oxidase and Endothelial Nitric Oxide Synthase,” Circulation 105: 1656–62 (2002).(j)Haffner et al., “Mortality From Coronary Heart Disease in Subjects With Type 2 Diabetes and in Nondiabetic Subjects With and Without Prior Myocardial Infarction,” N Engl J Med 339: 229–34 (1998).(k)Hiller et al., “Diabetic Retinopathy and Cardiovascular Disease in Type II Diabetics. The Framingham Heart Study and the Framingham Eye Study,” Am J Epidemiol 128: 402–9 (1988).(l)Jude et al., ‘Peripheral Arterial Disease in Diabetic and Nondiabetic Patients: a Comparison of Severity and Outcome,” Diabetes Care 24: 1433-7 (2001).(m)Kip et al., “Coronary Angioplasty in Diabetic Patients. The National Heart, Lung, and Blood Institute Percutaneous Transluminal Coronary Angioplasty Registry,” Circulation 94: 1818–25 (1996)(n)Lerman et al., “Cellular Dysfunction in the Diabetic Fibroblast: Impairment in Migration, Vascular Endothelial Growth Factor Production, and Response to Hypoxia,” Am J Pathol 162: 303–12 (2003).(o)Marsh et al., “Hypoxic Induction of Vascular Endothelial Growth Factor is Markedly Decreased in Diabetic Individuals Who Do Not Develop Retinopathy,” Diabetes Care 23: 1375–80 (2000).(p)Partamian et al., “Acute Myocardial Infarction in 258 Cases of Diabetes. Immediate Mortality and Five-Year Survival.” N Engl J Med 273: 455–61 (1965).(q)Rivard et al., “Rescue of Diabetes-Related Impairment of Angiogenesis By Intramuscular Gene Therapy With Adeno-VEGF,” Am J Pathol 154: 355–63 (1999)(r)Schatteman et al., “Blood-Derived Angioblasts Accelerate Blood-Flow Restoration in Diabetic Mice,” J Clin Invest 106: 571–8 (2000).(s)Tepper et al., “Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures,” Circulation 106: 2781–6 (2002).(t)Yarom et al., “Human Coronary Microvessels in Diabetes and Ischaemia. Morphometric Study of Autopsy Material,” JPathol 166: 265–70 (1992).Despite the preponderance of these observations, the mechanisms underlying impaired neovascularization in diabetes remain unclear. Impaired VEGF expression has been implicated as a significant contributing factor (Rivard et al., “Rescue of Diabetes-Related Impairment of Angiogenesis By Intramuscular Gene Therapy With Adeno-VEGF,” Am J Pathol 154:355-63 (1999); Schratzberger, et al., “Reversal of Experimental Diabetic Neuropathy by VEGF Gene Transfer,” J Clin Invest 107:108392 (2001); Aiello et al., “Role of Vascular Endothelial Growth Factor in Diabetic Vascular Complications,” Kidney Int Suppl 77:S113-9 (2000)). A detailed understanding of the mechanism of reduced VEGF expression would provide a useful framework for new approaches to improve diabetic outcomes following ischemic events.Ischemia-Induced Neovascularization Occurs by Two Mechanisms: Angiogenesis and Vasculogenesis.
After the appropriate hypoxic signaling cascade is initiated, compensatory vascular growth in response to ischemic insult occurs by two different mechanisms (FIG. 1). In angiogenesis, mature resident endothelial cells proliferate and sprout new vessels from an existing vessel in response to an angiogenic stimulus. In a more recently described mechanism, termed vasculogenesis, circulating cells with characteristics of vascular stem cells (endothelial progenitor cells, or EPCs) are mobilized from the bone marrow in response to an ischemic event, and then home specifically to ischemic vascular beds and contribute to neovascularization (Asahara et al., “Isolation of Putative Progenitor Endothelial Cells for Angiogenesis,” Science 275:964-7 (1997); Shi et al., “Evidence for Circulating Bone Marrow-Derived Endothelial Cells,” Blood 92:362-7 (1998); Asahara et al., “Bone Marrow Origin of Endothelial Progenitor Cells Responsible for Postnatal Vasculogenesis in Physiological and Pathological Neovascularization,” Circ Res 85:221-8 (1999); Isner et al., “Angiogenesis and Vasculogenesis as Therapeutic Strategies for Postnatal Neovascularization,” J Clin Invest 103:1231-6 (1999); Crosby et al., “Endothelial Cells of Hematopoietic Origin Make a Significant Contribution to Adult Blood Vessel Formation,” Circ Res 87:728-30 (2000); Pelosi et al., “Identification of the Hemangioblast in Postnatal Life,” Blood 100:3203-8 (2002)).
Hypoxia-Inducible Factor-1 (HIF-1) is the Central Mediator of the Hypoxia Response Including Subsequent Blood Vessel Growth.
The observation that ischemia regulates blood vessel growth has been known for many years, yet the responsible factor eluded identification until 1992, when Semenza and colleagues described a hypoxia-responsive transcription factor (HIF-1) which mediates erythropoietin gene upregulation (Semenza et al., “A Nuclear Factor Induced by Hypoxia via de Novo Protein Synthesis Binds to the Human Erythropoietin Gene Enhancer at a Site Required for Transcriptional Activation,” Mol Cell Biol 12:5447-54 (1992); Semenza et al., “Hypoxia-Inducible Nuclear Factors Bind to an Enhancer Element Located 3′ to the Human Erythropoietin Gene,” Proc Natl Acad Sci USA 88:5680-4 (1991)). HIF-1 proved to be a novel transcription factor conserved in all metazoan phyla and is ubiquitously present in all cells examined thus far (Carmeliet et al., “Abnormal Blood Vessel Development and Lethality in Embryos Lacking a Single VEGF Allele,” Nature 380:435-9 (1996)). Evidence for its involvement in angiogenesis stemmed from the initial observation that VEGF was strongly upregulated by hypoxic conditions (Shweiki et al., “Vascular Endothelial Growth Factor Induced by Hypoxia May Mediate Hypoxia-Initiated Angiogenesis,” Nature 359:843-5 (1992)). Soon thereafter, HIF-1 was shown to be the transcription factor responsible for VEGF upregulation by hypoxia and hypoglycemia (Forsythe et al., “Activation of Vascular Endothelial Growth Factor, Gene Transcription by Hypoxia-Inducible Factor 1,” Mol Cell Biol 16:4604-13 (1996)). It is now clear that HIF-regulated VEGF expression is essential for vascular development during both embryogenesis and postnatal neovascularization in physiologic and pathologic states (Carmeliet et al., “Abnormal Blood Vessel Development and Lethality in Embryos Lacking a Single VEGF Allele,” Nature 380:435-9 (1996); Carmeliet et al., “Abnormal Blood Vessel Development and Lethality in Embryos Lacking a Single VEGF Allele,” Nature 380:435-9 (1996); Iyer et al., “Cellular and Developmental Control of O2 Homeostasis by Hypoxia-Inducible Factor I Alpha,” Genes Dev 12:149-62 (1998)). HIF-1 consists of the oxygen-regulated HIF-1α subunit and the HIF-1β subunit, which is not regulated by oxygen. HIF-1 is now believed to be the master transcription factor directing the physiologic response to hypoxia by upregulating pathways essential for adaptation to ischemia, including angiogenesis, vasculogenesis, erythropoiesis and glucose metabolism (FIG. 2).
Regulation of HIF-1α Transcriptional Activation.
The HIF-1 transcriptional complex is comprised of HIF-1α/β and more than seven other factors that modulate gene transcription. The two predominant functional components of this complex are HIF-1α and CBP/p300, which directly interact to transactivate gene expression. HIF-1α function is predominantly regulated by oxygen via protein stabilization and post-translational modification. Recent reports demonstrate that HIF-1α is activated by phosphorylation in vitro, enhancing HIF-mediated gene expression (Richard et al., “p42/p44 Mitogen-Activated Protein Kinases Phosphorylate Hypoxia-Inducible Factor 1 alpha (HIF-1 alpha) and Enhance the Transcriptional activity of HIF-1,” J Biol Chem 274:32631-7(1999)). Whether this modification results in a direct stimulation of the transactivation function of HIF-1α itself or facilitates recruitment of co-activators is not clear (Richard et al., “p42/p44 Mitogen-Activated Protein Kinases Phosphorylate Hypoxia-Inducible Factor I alpha (HIF-1 alpha) and Enhance the Transcriptional activity of HIF-1,” J Biol Chem 274:32631-7 (1999); Sang et al., “Signaling Up-Regulates the Activity of Hypoxia-Inducible Factors by Its Effects on p300,” J Biol Chem 278:14013-9 (2003)).
It has also been recently demonstrated that CBP/p300 also undergoes phosphorylation in vitro, enhancing its ability to function as a transcriptional activator in association with HIF-1α (Sang et al., “Signaling Up-Regulates the Activity of Hypoxia-Inducible Factors by Its Effects on p300,” J Biol Chem 278:14013-9 (2003)). Thus, cellular states that promote phosphorylation of these two factors likely increase hypoxia-induced gene expression, while those that favor dephosphorylation have the opposite effect. Although HIF-1 mediated gene expression is essential for both angiogenesis and vasculogenesis, the role of its regulation in diabetic states has not been previously examined.
Both Angiogenesis and Vasculogenesis are Modulated by VEGF.
It is well known that angiogenesis is mediated by VEGF and this mechanism has been extensively investigated (Ferrara et al., “The Biology of VEGF and its Receptors,” Nat Med 9:669-76 (2003)). Recently, VEGF has also been implicated in regulation of vasculogenesis (FIG. 2). Ischemia is a potent mobilizer of endothelial progenitor cells from the bone marrow. This appears to be mediated through VEGF signaling, as EPCs express both VEGF receptor I and 2 on their cell surface (Asahara et al., “VEGF Contributes to Postnatal Neovascularization by Mobilizing Bone Marrow-Derived Endothelial Progenitor Cells,” Embo J 18:3964-72 (1999); Takahashi et al., “Ischemia- and Cytokine-Induced Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells for Neovascularization,” Nat Med 5:434-8 (1999); Kalka et al., “Vascular Endothelial Growth Factor(165) Gene Transfer Augments Circulating Endothelial Progenitor Cells in Human Subjects,” Circ Res 86:1198-202 (2000); Gill et al., “Vascular Trauma Induces Rapid but Transient Mobilization of VEGFR2(+)AC133(+) Endothelial Precursor Cells,” Circ Res 88:167-74 (2001); Hattori et al., “Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment of Vasculogenic and Hematopoietic Stem Cells,” J Exp Med 193:1005-14 (2001)). Given that VEGF production is impaired in diabetes mellitus, it seems likely that various aspects of vasculogenesis, including EPC mobilization, may also be impaired. Indeed, recent evidence has demonstrated that the incorporation of these vascular progenitors into blood vessels is decreased in diabetic states.
VEGF Expression may be Regulated in a Tissue-Specific Manner.
It also clear that various tissues and organs in diabetic patients exhibit different pathologies. The retina is often characterized by excessive angiogenesis, while skin, muscle, and nerves in diabetic patients suffer from a paucity of new vessel formation. Similarily, diabetic retinopathy has been characterized by increased levels of ocular VEGF levels, (Aiello et al., “Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders,” N Engl J Med 331:1480-7 (1994); Adamis et al., “Increased Vascular Endothelial Growth Factor Levels in the Vitreous of Eyes with Proliferative Diabetic Retinopathy,” Am J Ophthalmol 118:445-50 (1994)), while impaired wound healing has been characterized by severely decreased levels of VEGF (Frank et al., “Regulation of Vascular Endothelial Growth Factor Expression in Cultured Keratinocytes. Implications for Normal and Impaired Wound Healing,” J Biol Chem 270:12607-13 (1995); Peters et al., “Vascular Endothelial Growth Factor Receptor Expression During Embryogenesis and Tissue Repair Suggests a Role in Endothelial Differentiation and Blood Vessel Growth,” Proc Natl Acad Sci USA 90:8915-9 (1993); Silhi, N., “Diabetes and Wound Healing,” J Wound Care 7:47-51 (1998); Brown, L. F., “Expression of Vascular Permeability Factor (Vascular Endothelial Growth Factor) by Epidermal Keratinocytes During Wound Healing,” J Exp Med 176:1375-9 (1992); Nissen et al., “Vascular Endothelial Growth Factor Mediates Angiogenic Activity During the Proliferative Phase of Wound Healing,” Am J Pathol 152:1445-52 (1998)). This so-called “diabetic paradox,” by which the diabetic phenotype exhibits both excessive and impaired new blood vessel formation in different tissues, leads to different types of complications. It is believed this phenomenon represents a cell- and tissue-specific difference in the transcriptional regulation of VEGF.
Hyperglycemia Results in Specific Impairments of Cellular Function Through Overproduction of Reactive Oxygen Species: a Potential Link to VEGF.
The cellular mechanism that accounts for impaired hypoxia-induced VEGF and SDF-1 expression has not yet been determined. Recently, the biochemical basis for hyperglycemia-induced cellular damage was described, demonstrating that many of the effects of high glucose are mediated through four specific cellular pathways (FIG. 3) (Brownlee, “Biochemistry and Molecular Cell Biology of Diabetic Complications,” Nature 414:813-20 (2001); Nishikawa et al., “Normalizing Mitochondrial Superoxide Production Blocks Three Pathways of Hyperglycaemic Damage,” Nature 404:787-90 (2000)). Intracellular elevations in glucose increase flux of metabolites through glycolysis and the Kreb's cycle, resulting in overproduction of ROS by the mitochondria. Overproduction of ROS inhibits GAPDH activity, resulting in accumulation of early glucose metabolites in the initial phases of glycolysis. The abundance of these metabolites and their inability to progress through glycolysis causes shunting of these intermediates into alternative pathways of glucose utilization (polyol pathway, hexosamine pathway, protein kinase C pathway, and AGE pathway, FIG. 3). Accumulation of end products in each of these pathways leads to specific changes in cellular function, including gene expression (Nissen et al., “Vascular Endothelial Growth Factor Mediates Angiogenic Activity During the Proliferative Phase of Wound Healing,” Am J Pathol 152:1445-52 (1998)), and are implicated in the pathophysiology of diabetic complications (Brownlee, “Biochemistry and Molecular Cell Biology of Diabetic Complications,” Nature 414:813-20 (2001)). Indeed, specific blockade of one, several, or all of these pathways has been shown to prevent diabetic complications in an animal model, including those complications that result from ischemic injury (Hammes et al., “Benfotiamine Blocks Three Major Pathways of Hyperglycemic Damage and Prevents Experimental Diabetic Retinopathy,” Nat Med 9:294-9 (2003); Obrosova et al., “Aldose Reductase Inhibitor Fidarestat Prevents Retinal Oxidative Stress and Vascular Endothelial Growth Factor Overexpression in Streptozotocin-Diabetic Rats,” Diabetes 52:864-71 (2003)).
Hyperglycemia-induced reactive oxygen species also impair the ability of HIF-1α to mediate appropriate upregulation of VEGF and the chemokine SDF-1 that are required for neovascularization in ischemic settings. This impairment also affects hypoxia-specific functions of vascular effector cells. This results in impaired angiogenesis, vasculogenesis, and diminished tissue survival in diabetic states. Increased free fatty acid flux has been shown to increase ROS by identical mechanisms (Du et al., “Insulin Resistance Causes Proatherogenic Changes in Arterial Endothelium by Increasing Fatty Acid Oxidation-Induced Superoxide Production” J. Clin. Invest. in press).
The present invention is directed to treating or preventing the pathologic sequelae of acute hyperglycemia and/or increased fatty acid flux in a subject, thus, preventing metabolite-induced reactive oxygen-species mediated injury.